The high catalytic rate of the cold-active Vibrio alkaline phosphatase requires a hydrogen bonding network involving a large interface loop.

FEBS Open Bio
Jens Guðmundur HjörleifssonBjarni Ásgeirsson

Abstract

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability. Here, we designed several different variants of VAP with the aim of removing intersubunit interactions at the dimer interface. Breaking the intersubunit contacts from one residue in particular (Arg336) reduced the temperature stability of the catalytically potent conformation and caused a 40% drop in catalytic rate. The high catalytic rates of enzymes from cold-adapted organisms are often associated with increased dynamic flexibility. Comparison of the relative B-factors of the R336L crystal structure to that of the wild-type confirmed surface flexibility was increased in a loop on the opposite monomer, but not in the large loop. The increase in flexibi...Continue Reading

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Methods Mentioned

BETA
circular dichroism
NMR

Software Mentioned

refmac
graphpad prism
coot
cpp
kaleidagraph
molrep

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